1. Field of the Invention
The present invention relates to signals or molecular species involved in senescence and particularly, to signals or molecular species involved in cellular senescence and their use.
2. Description of the Related Art
Cellular senescence plays an important role in complex biological processes, including development, aging, and tumorigenesis, and many attempts have been made to understand some of its fundamental features (Peacocke and Campisi, 1991; Smith and Pereira-Smith, 1996). One of the hallmarks of cellular senescence is the hyporesponsiveness to the growth factors and mitogens.
Age-related quantitative and qualitative alterations in growth factor receptors may account, at least in part, for the diminished responsiveness of senescent cells to growth factors. The decreased numbers of high and low affinity epidermal growth factor (EGF) receptors during serial cultivation in vitro have been reported in human omental microvascular endothelial cells (Matsuda et al., 1992), and the diminished responsiveness to the EGF of particular chondrocytes derived from old animals also seems to be ascribed to a reduction in the number of EGF receptors (Ribault et al., 1998). Age-related reductions in the numbers of PDGF (platelet-derived growth factor) binding sites or PDGF receptors have also been demonstrated in several cell systems including human smooth muscle cells (Mori et al., 1993; Aoyagi et al., 1995).
In contrast, some senescent cells in culture retain functional growth factor receptor systems and have normal numbers of receptors for growth factors with normal binding affinity (Paulsson et al., 1986; Gerhard et al., 1991; Hensler and Pereira-Smith, 1995; Park et al., 2000). In these cases, post-receptor signal transduction pathways in cellular senescence could be responsible for the attenuated responsiveness to growth factors. Upregulation of caveolin and/or the down-regulation of amphiphysin seems to account for the unresponsiveness of senescent fibroblasts to EGF stimulation (Park et al., 2000, 2001). Defects in calcium-phospholipid-dependent protein kinase C (PKC) pathway have also been suggested to be involved in the mitogenic defect of senescent cells (Pascale et al., 1998; Venable and Obeid, 1999). Since phosphatidylcholine-specific phospholipase D (PLD) is activated by PKC and followed by the action of phosphatidic acid, phosphohydrolase results in delayed and more sustained diacylglycerol formation, which might be responsible for the sustained activation of PKC, the activity of PLD could be altered during the aging process. Indeed, Venable et al. (1994) demonstrated a defect in the serum-stimulated PLD/PKC pathway during cellular senescence.
In contrast to previous reports demonstrating a decrease in agonist-stimulated signaling events, some reports have shown an increase in the signaling events. Bradykinin increases IP3 formation in fibroblasts from normal aged and Alzheimer donors (Huang et al., 1991) and PLD activity in human senescent fibroblasts (Meacci et al., 1995) more so than in young counterparts. Human lymphocytes showed elevated mitogen-induced Ca2+ responses after exposure to beta-amyloid, the main component of senile plaques in Alzheimer disease (Eckert et al., 1994). These results suggest that the effect of aging on signaling events could be agonist-specific.
PDGF transfers a mitogenic signal via a plasma membrane-bound receptor possessing the activity of protein tyrosine kinase, while lysophosphatidic acid (LPA) acts as an extracellular messenger through guanine nucleotide binding protein (G-protein). Since both PDGF and LPA elicit the same signaling events, including the mobilization of intracellular Ca2+, actin polymerization and phosphatidic acid production in human diploid fibroblasts, we compared the responsiveness of senescent or near-senescent cells to two different major mitogenic agonists, PDGF and LPA.
Meanwhile, lysophosphatidic acid (LPA) is a lipid mediator with diverse biological activities, including changes in cell shape, chemotaxis, proliferation, and differentiation (Moolenaar et al., 1997; An et al., 1998c). LPA is generated by phospholipase cleavage of membrane phospholipids from stimulated cells, especially activated platelets (Gaits et al., 1997). The intracellular biochemical signaling events that mediate the effects of LPA include increases in cytoplasmic calcium concentration, stimulation of phospholipases, activation of phosphatidylinositol 3-kinase, the Ras-Raf-MAP kinase cascade, and inhibition of adenylyl cyclase (AC) (Moolenaar et al., 1997). Recently, cell surface G-protein-coupled receptors for LPA were identified as a family of endothelial cell differentiation genes (EDGs) (LPA receptors reviewed in Contos et al., 2000; Fukushima et al., 2001). The major members of the EDG family interacting with LPA were shown to be EDG-2 (Hecht et al., 1996), EDG-4 (An et al., 1998a), and EDG-7 (Bandoh et al., 1999). LPA is also a low affinity agonist for EDG-1 (Lee et al., 1998).
Specific response of the G-protein-coupled receptor to a ligand might be possible only when appropriate G-proteins are coupled to the receptor (Figler et al., 1996; Moolenaar, 1997). EDG-2 is coupled to pertussis toxin-sensitive Gi, whereas EDG4 is coupled to both Gi and Gq (An et al., 1998), and EDG7 to a pertussis toxin-insensitive G-protein(s), possibly Gq (Bandoh et al., 1999; Im et al., 2000). Gq protein could mediate inositol 1,4,5-trisphosphates (IP3) production and subsequent Ca++ mobilization, whereas Gi could mediate the inhibition of AC. This complex linkage of LPA-signaling system with a variety of factors suggests the possibility of functional deterioration in age-dependent manner.
Cellular cAMP can be synthesized by activated AC and hydrolyzed by the cyclic nucleotide phosphodiesterases (PDE). An increase in the cAMP content results in the activation of cAMP-dependent protein kinase (PKA), which phosphorylates cellular proteins, regulates gene expression by activating cAMP response element binding protein (CREB), and thus results in numerous cellular responses including cell proliferation, differentiation, metabolism, and neuronal functions (Taussig and Gilman, 1995).
Previously, it was reported that PGE1-induced cAMP accumulation and subsequent phosphorylation of CREB by protein kinase A was markedly attenuated in senescent cells (Chin et al., 1996). However, cAMP signaling induced by forskolin or interferon-γ-inducible protein-10 (IP-10), is relatively maintained in senescent human diploid fibroblasts Hs68 (Shiraha et al., 2000). Rather enhanced cAMP stimulation was observed in late passage-human embryonic lung fibroblasts treated with serum (Polgar et al., 1978) and in senescent IMR-90 lung fibroblasts treated with isoproterenol (Ethier et al., 1992). While the response in VSMC cultured from the older rats was actually increased compared to the VSMC cultured from the younger rats, there was a reduction of cAMP response to isoproterenol in fibroblasts cultured from the older rats (Chin and Hoffman, 1991). These results suggest that the effect of aging on cAMP signaling events could be agonist- and cell-specific.
The patent applications related to nucleic acid and proteins associated with aging process, disclosed in WO 99/52929 and WO 01/23615.
As described above, a variety of theories have been proposed, there remains a need of more evident elucidation for cellular senescence, a need of specific biomarker for identifying senescent cell, and a need of biomolecule for modulating cellular senescence.
In particular, the prospect of reversing senescence and restoring normal physiological function has an importance in certain diseases associated with senescence, for example, Werner Syndrome and Hutchinson-Gilford Syndrome.
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.